We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human α3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express α3, α5, α7, β2, and β4 AChR subunits. From these subunits, several types of α3 AChRs as well as homomeric α7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect α3β4 AChRs. α7 AChR function was not detected, yet we estimate that there are 70% as many surface α7 AChRs in IMR-32 when compared with α3 AChRs. Agonist potencies (EC₅₀ values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16±1 μM) > nicotine (Nic; 48 ± 7 μM) ≥ cytisine (Cyt; 57 ± 3 μM) = acetylcholine (ACh; 59 ± 6 μM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s⁻¹ (300 μM ACh; −60 mV). Assays that used mAbs confirmed the predominance of α3- and β4-containing AChRs in IMR-32 cells. Although 18% of total α3 AChRs contained β2 subunits, no β2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface α3β2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in α3β2 transfected HEK cells. Characterization of various α3 AChRs expressed in HEK cell lines revealed that the functional properties of the α3β4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC₅₀ values) for this line was DMPP (14 ± 1 μM) = Cyt (18 ± 1 μM) > Nic (56 ± 15 μM > ACh (79 ± 8 μM). The efficacies of both Cyt and DMPP were ∼80% when compared with ACh and the desensitization rate was 2 s⁻¹. These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to α3β4 AChRs.